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Genomics & Epigenetics

ChIP-seq Workflows Run Best with Automated DNA Size Selection

Chromatin immunoprecipitation sequencing, better known as ChIP-seq, is a massively parallel approach for understanding the interactions between proteins and DNA. This is especially important for determining the activity of transcription factors, which is why it’s frequently used to learn about the complicated series of biological steps leading to cancer. It’s also key to many epigenetic…

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Generating High-Quality Genome Assemblies from Metagenomic Sequencing

The decreasing costs in genomic sequencing over the past decade have inspired researchers to apply shotgun next-generation sequencing to entire microbial communities. While the reads generated typically cannot be assembled cleanly into individual genomes, there is often enough information produced to identify most microbes present in the population. However, this approach lacks sufficient resolution to…

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Get to Know Your Reference Genome (GRCh37 vs GRCh38)

Whether your experiment relies upon a reference-based genome assembly or mapping reads to a reference genome to identify variants, you need to choose a human reference genome assembly. But wait! You go to the FTP site of NCBI’s refseq and click on the Homo sapiens folder. There you are presented with two choices. Which one…

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CRISPR-Inspired Method Targets Large, Repetitive DNA Elements

Target capture through PCR has been a mainstay in genomics for years, but scientists working on especially repetitive, poorly characterized, or rapidly evolving regions continue to struggle to fish out those stretches of DNA for further study. However, whole genome sequencing, the only other alternative for these regions, can force researchers to pay for much…

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De Novo DNA Sequencing and the Special k-mer

The technology for DNA sequencing was developed back in 1977 thanks to Frederick Sanger. It took a bit longer before it was possible to sequence a complete genome. This is because we needed an appropriate mathematical model and massive computational power to assemble millions or billions of small reads to a larger complete genome. Today’s…

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How to Become a Bioinformatician

If you want to start a journey towards being a bioinformatician, you must first know the meaning of the term bioinformatics. You will also need to know what bioinformaticians do, and what you need to know to land your first entry level job as a bioinformatician. Most bioinformaticians work within different medical science and health…

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Using dbSNP and ClinVar to Classify Gene Variants

As we discussed previously, the gaps in our understanding of the human genome make variant classification an extremely difficult job. However, with each passing day our knowledge increases, and the tools to help us become increasingly more efficient. Let’s pick up where we left off in our first article about variants. After checking Ensemble to…

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A Crash Course in BLAST Searching

Simple BLAST searching is pretty straightforward to many of us. Just plug in your sequence, select the species genome, and hit search! But have you ever wondered what it takes to run a BLAST query using these mammoth-sized (no pun intended!) sequence databases? BLAST searching can produce dozens, hundreds, or even thousands of candidate alignments.…

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How to Use Ensembl to Classify Gene Variants

Even though our knowledge about genomes grows daily, and in huge leaps, we sometimes need to remind ourselves that DNA was first isolated in 1869 and its molecular structure was only identified in 1953. The PCR reaction only hit the scientific community as recently as 1983! So even though we are growing fast, we are…

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Next Gen Sequencing Challenges

Next generation sequencing opened the doors to our genome. It gives massive amounts of information in a week – whereas Sanger sequencing takes thrice as long, and causes lab lesions due to the abusive use of pipettes. Indeed, with minimal hands-on procedures we obtain a lot of data. But nothing in Science is ever easy.…

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DNA Extraction from FFPE Tissues for NextGen Sequencing

Rapid genomic analysis offered by next generation sequencing (NGS) is ideal for personalized medicine approaches to clinical genetics, microbiological profiling, and diagnostic oncology. Many standard clinical samples are preserved as formalin-fixed, paraffin-embedded (FFPE) tissues, which presents obstacles for use in NGS analysis. FFPE tissue preservation has the benefit of keeping samples intact for histological examination…

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Best Practices for DNA Shearing for NGS

Construction of high-quality sequencing libraries is pivotal to successful NGS, and DNA quality is one of the most critical aspects of library preparation. As this Nature Methods paper illustrates, DNA shearing involves appropriate and consistent fragment sizes for sensitive and accurate sequencing, and the fragments must be accurately analyzed prior to sequencing to measure molarity…

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Codon Optimization 101

The intriguing thing about protein expression is that the combination of transfer RNAs (tRNAs) that translate the 3 letter codon into an amino acid (aa) far exceeds the number of existing amino acids (aa). If you do the math correctly, the maximum number of unique combinations using the triplet code to code for the 4…

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Maxam-Gilbert Sequencing: What Was It, and Why It Isn’t Anymore

In the mid-1970s, two methods were developed for directly sequencing DNA: the Maxam-Gilbert sequencing (or chemical sequencing) method and the Sanger chain-termination method. Indeed, in 1980, both Walter Gilbert and Frederick Sanger were awarded The Nobel Prize in Chemistry for “their contributions concerning the determination of base sequences in nucleic acids”. Actually, each got a…

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An Introduction to Nanopore Sequencing

DNA sequencing is the most powerful method to reveal genetic variations at the molecular level, leading to a better understanding of our body in physiological settings, and pathological conditions. It is the beginning of the long road towards better diagnostics and personalized medicine. Even though there have been great advances in DNA sequencing technologies there…

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A Crash Course in CRISPR-Cas9 Editing in Drosophila

CRISPR-Cas9 has become a magic tool for molecular biologists, transforming genetic engineering from a once unbelievable dream into tangible reality. Today, you can easily edit primary cells or cell lines within a few weeks with well-established protocols or others’ hands-on advice. However, no matter how many success stories you find online or hear at seminars…

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How to Design a CRISPR Experiment and Start Genome Editing

CRISPR and the CRISPR Associated system (Cas) are powerful gene editing technologies. Originally identified and characterized in bacteria, the endogenous CRISPR systems act as an RNA-based defense mechanism against invading phage DNA. CRISPR cas9 gene editing was adapted for genome editing in 2013 and has since been exploited for its ability to generate targeted double-stranded DNA…

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